Next-Generation Sequencing

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.title[
# Next-Generation Sequencing
]
.author[
### <b>Ratnesh Bhai Mehta</b> • 28-Apr-2023
]
.institute[
### Zifo RnD Solutions
]

---

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## Sequencing Technologies

<br>

### Sequencing: Definition

<br>

* In [genetics](https://en.wikipedia.org/wiki/Genetics) and [biochemistry](https://en.wikipedia.org/wiki/Biochemistry), [**sequencing**]((https://en.wikipedia.org/wiki/Sequencing)) means to determine the [primary structure](https://en.wikipedia.org/wiki/Biochemistry) (sometimes incorrectly called the primary sequence) of an unbranched [biopolymer](https://en.wikipedia.org/wiki/Biopolymer).

* Types
  
  * Principles
  
  * Their “+” and “-”

<br>

---

## Sequencing Technologies

<br>

### Technologies

<br>

<table>
 <thead>
  <tr>
   <th style="text-align:left;"> Company </th>
   <th style="text-align:left;"> Platform </th>
   <th style="text-align:left;"> Amplification </th>
   <th style="text-align:left;"> Sequencing.Method </th>
  </tr>
 </thead>
<tbody>
  <tr>
   <td style="text-align:left;"> Illumina </td>
   <td style="text-align:left;"> MiSeq, NextSeq, NovaSeq </td>
   <td style="text-align:left;"> Bridge PCR </td>
   <td style="text-align:left;"> Synthesis (SBS) </td>
  </tr>
  <tr>
   <td style="text-align:left;"> ThermoFisher </td>
   <td style="text-align:left;"> Ion Torrent </td>
   <td style="text-align:left;"> emPCR </td>
   <td style="text-align:left;"> Synthesis (pH) </td>
  </tr>
  <tr>
   <td style="text-align:left;"> Pacific Biosciences </td>
   <td style="text-align:left;"> Sequel II </td>
   <td style="text-align:left;"> None </td>
   <td style="text-align:left;"> Synthesis </td>
  </tr>
  <tr>
   <td style="text-align:left;"> Oxford Nanopore Technologies </td>
   <td style="text-align:left;"> MinION, GridION, PromethION </td>
   <td style="text-align:left;"> None </td>
   <td style="text-align:left;"> Flow </td>
  </tr>
</tbody>
</table>

---

## Sequencing Technologies

<br>

### Differences Between Platforms

<br>

* Technology : Chemistry + Signal Detection

* Run Time : Varies from hours to days

* Product Range : Mb – Gb

* Read Length : < 100 bp to >20 Kbp

* Accuracy per base: 0.1 % to 15%

* Cost per base: Varies

---

## Sequencing Technologies

<br>

### Illumina

<table>
 <thead>
  <tr>
   <th style="text-align:left;"> Instrument </th>
   <th style="text-align:left;"> Yield...Runtime.. </th>
   <th style="text-align:left;"> Read.Length </th>
   <th style="text-align:left;"> Error.Rate.. </th>
   <th style="text-align:left;"> Error.Type </th>
  </tr>
 </thead>
<tbody>
  <tr>
   <td style="text-align:left;"> MiSeq </td>
   <td style="text-align:left;"> 15 Gb, 4-55 hrs </td>
   <td style="text-align:left;"> 2 X 300 bp </td>
   <td style="text-align:left;"> 0.47 </td>
   <td style="text-align:left;"> Substitution </td>
  </tr>
  <tr>
   <td style="text-align:left;"> NextSeq </td>
   <td style="text-align:left;"> 330 Gb, 11-48 hrs </td>
   <td style="text-align:left;"> 2 X 150 bp </td>
   <td style="text-align:left;"> 0.59 </td>
   <td style="text-align:left;"> Substitution </td>
  </tr>
  <tr>
   <td style="text-align:left;"> NovaSeq </td>
   <td style="text-align:left;"> 6000 Gb, 13-44 hrs </td>
   <td style="text-align:left;"> 2 X 250 bp </td>
   <td style="text-align:left;"> 0.11 </td>
   <td style="text-align:left;"> Substitution </td>
  </tr>
</tbody>
</table>

[*https://sapac.illumina.com/systems/sequencing-platforms.html](https://sapac.illumina.com/systems/sequencing-platforms.html)
[#https://academic.oup.com/nargab/article/3/1/lqab019/6193612](https://academic.oup.com/nargab/article/3/1/lqab019/6193612)

<br>

#### Main Application

* Whole Genome, Whole Exome, Targeted Panel Sequencing

* Transcriptome Sequencing (total RNA-Seq, mRNA-Seq, gene expression profiling, miRNA)

* Single-Cell Profiling (scRNA-Seq, snDNA-Seq, scDNA-Seq, oligo tagging assays)

* Methylome and ChiPSeq

* Metagenomic Profiling (16S RNA, shotgun metagenomics, metatranscriptomics)

---

## Sequencing Technologies

<br>

### ILLUMINA: Sequencing By Synthesis (SBS)

---

## Sequencing Technologies

<br>

### Thermofisher

<br>

<table>
 <thead>
  <tr>
   <th style="text-align:left;"> Instrument </th>
   <th style="text-align:left;"> Yield...Runtime </th>
   <th style="text-align:left;"> Read.Length </th>
   <th style="text-align:left;"> Error.Rate. </th>
   <th style="text-align:left;"> Error.Type </th>
  </tr>
 </thead>
<tbody>
  <tr>
   <td style="text-align:left;"> Ion Torrent Genexus System </td>
   <td style="text-align:left;"> Ion Torrent Genexus System </td>
   <td style="text-align:left;"> 200-600 bp </td>
   <td style="text-align:left;"> 1.50% </td>
   <td style="text-align:left;"> InDel </td>
  </tr>
</tbody>
</table>

[#https://www.nature.com/articles/s41598-017-08139-y](https://www.nature.com/articles/s41598-017-08139-y)

<br>

* Main Application
   * Small Genomes, Whole Exome, Targeted Panel Sequencing
   
   * Transcriptome Sequencing (total RNA-Seq, mRNA-Seq, gene expression profiling,miRNA)
   
   * Metagenomic Profiling (16S RNA)

---

## Sequencing Technologies

<br>

### Ion Semiconductor Sequencing (pH)

---

## Sequencing Technologies

<br>

### Pacific Biosciences

<br>

<table>
 <thead>
  <tr>
   <th style="text-align:left;"> Instrument </th>
   <th style="text-align:left;"> Yield...Runtime </th>
   <th style="text-align:left;"> Read.Length </th>
   <th style="text-align:left;"> Error.Rate </th>
   <th style="text-align:left;"> Error.Type </th>
  </tr>
 </thead>
<tbody>
  <tr>
   <td style="text-align:left;"> Sequel II </td>
   <td style="text-align:left;"> 250-450 Gb, ~30hrs </td>
   <td style="text-align:left;"> ~15-20 Kbp </td>
   <td style="text-align:left;"> 13-15% </td>
   <td style="text-align:left;"> Mismatches </td>
  </tr>
</tbody>
</table>

[#https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5553090/](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5553090/)

<br>

* Main Application
  * Large Genomes
  
  * Transcriptome Sequencing (total RNA-Seq, mRNA-Seq)
  
  * Metagenomic Profiling (Shotgun Sequencing)

---

## Sequencing Technologies

<br>

### Single Molecule Real Time Sequencing (SMRT)

---

## Sequencing Technologies

<br>

### Oxford Nanopore Technologies

<br>

<table>
 <thead>
  <tr>
   <th style="text-align:left;"> Instrument </th>
   <th style="text-align:left;"> Yield...Runtime </th>
   <th style="text-align:left;"> Read.Length.. </th>
   <th style="text-align:left;"> Error.Rate.. </th>
   <th style="text-align:left;"> Error.Type </th>
  </tr>
 </thead>
<tbody>
  <tr>
   <td style="text-align:left;"> MinION </td>
   <td style="text-align:left;"> 10-50 Gb, ~72 hrs </td>
   <td style="text-align:left;"> ~882 Kbp </td>
   <td style="text-align:left;"> ~30% </td>
   <td style="text-align:left;"> Mismatches </td>
  </tr>
</tbody>
</table>

[*https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5889714/](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5889714/)
[#https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5553090/](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5553090/)

<br>

* Main Application
  * Large Genomes, Targeted Sequencing
  
  * Transcriptome Sequencing (total RNA-Seq, mRNA-Seq)
  
  * Metagenomic Profiling (Shotgun Sequencing)
  
  * Epigenetics

---

## Sequencing Technologies

<br>

### Nanopore Sequencing

---

## Sequencing Technologies

<br>

### What is the best ?

<br>

* Design your experiment based on the scientific question.
  * Chose the best suited application for your project.
  
  * Find the most optimal sequencing technology.
  
  * Answer all questions about our technologies and applications, as well as 
    bioinformatics (down stream analysis)
---

## Sequencing Technologies

<br>

### Library Preparation for Sequencing

<br>

<img src="data:image/png;base64,#data/sequencing/libprep.jpg" width="100%" style="display: block; margin: auto;" />
---

## Sequence Database

<br>

### Sequencing Storage/ Cost

<br>

---

## Sequence Database

<br>

### Raw Data Archiving

<br>

---

## Sequence Database

<br>

### Sequencing Read Archive (SRA)

<br>

* The Sequence Read Archive (SRA) was created and engineered at the
  National Center for Biotechnology Information (NCBI), National Library of
  Medicine (NLM), National Institutes of Health (NIH), Department of Health
  and Human Services.
  
* The SRA is part of a cluster of sequencing data repositories called the "Trace
  Archives“ and is located under the "Primary Data Archives" at NCBI, which
  includes GenBank.
  
* The SRA is part of the International Nucleotide Sequence Database
  Collaboration (INSDC). The data model, data transfer protocols, and
  accession space are shared with the INSDC collaborators: European
  Bioinformatics Institute (EBI) and the DNA Data Bank of Japan (DDBJ).
---

## Sequence Database

<br>

### Primary Data Archive

<br>

---

## Sequence Database

<br>

### Primary Data Archive: Key Features

<br>

* Primary Data Archives are submitter-driven.

* Data Archive is different from file archive. It stores data not original files.

* Data Archive is not necessarily lossless. Some controlled loss of information
  or precision should be allowed.
  
* Internal storage format should be sufficiently uniform to enable validation,
  searching, sub-setting, etc...
  
* Extra effort is needed to support conversion from input formats as well as
  produce output formats. Large variety of formats significantly stresses
  archive’s resources.
  
* Additional benefit of conversion is that all data are validated before archival.

---

## Sequence Database

<br>

### NCBI-Sequence Read Archive (SRA): Walkthrough

---

## Sequence Database

<br>

### SRA: Download (SRA Toolkit)

---

## Sequence Database

<br>

### European Nucleotide Archive (ENA)

---

## Sequence Database

<br>

### DDBJ Sequence Read Archive (DRA)

---
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# Thank you. Questions?

.end-text[
<p class="smaller">
<span class="small" style="line-height: 1.2;">Graphics from </span><img src="./assets/freepik.jpg" style="max-height:20px; vertical-align:middle;"><br>
Created: 28-Apr-2023 • James Ashmore • <a href="https://www.zifornd.com/category/omics-bioinformatics">Bioinformatics</a> • <a href="https://www.zifornd.com">Zifo</a>
</p>
]